JPH02236454A - Automatic immunoassay device - Google Patents

Abstract

PURPOSE:To collect stable data by injecting a prescribed luminous auxiliary reagent into reaction tubes before injecting luminous reagents therein. CONSTITUTION:A tank 38 houses the auxiliary reagent and tank 39, 40 house the luminous reagent. The luminous auxiliary reagent, diluting liquid and luminous reagent are fed via a selector valve 33 and pumps 34 to 37. A buffer soln. housed in a tank 41 is used for the diluting liquid and cleaning liquid. The cleaning liquid is passed through a resistance tube 43, a preheater 44 and a valve 45 and is so controlled as to attain a stable prescribed temp. and flow rate, by which the reaction is facilitated and stabilized. The temp. at the time of a light emission reaction is stabilized with the luminous reagent as well by adding a heater to a mixer 46. The luminous reagents are simultaneously injected from respective bottles through a 3-way joint 42 and a mixer 46. Since the luminous reagents are mixed just prior to the execution of the detection in such a manner, the luminous reagents are maintained in the stable state.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to an immunoassay device that measures components contained in a sample such as serum or urine by an immune reaction using a solid-phase reagent. This invention relates to an automatic immunoassay device that injects a sample.

[Conventional technology]

Enzyme immunoassay is a method that uses enzyme activity as a marker to determine the extent of antigen-antibody reaction, and then quantifies the amount of antigen or antibody. Sandwich method that does not compete with antibodies (or antigens), competitive reaction methods that determine the amount of unlabeled antigen by competing enzyme-labeled antigens (or antibodies) with unlabeled antigens (or antibodies), and various other methods. There is.

FIG. 4 is a diagram for explaining the measurement principle by the sandwich method, and FIG. 5 is a diagram for explaining the measurement principle by the competitive method, in which 61 is a solid phase carrier, 62 is an antibody, 63 is an antigen to be measured, 64 is a labeled substance, 65 is a labeled antibody, 6B is a substrate,
67 indicates the product.

In the sandwich method, as shown in FIG. Add samples containing.

■ As a result, although there are various impurity components in the sample, the antibody 62 and antigen 63 react specifically (first reaction), and the antigen to be measured 63 binds to the immobilized antibody 62. , the impurities in the sample are discarded by washing.

(2) Next, an enzyme-labeled antibody 65 to which an enzyme is bound as a label θ4 is added.

(2) As a result, an antigen-antibody reaction (second reaction) occurs, and the enzyme-labeled antibody 65 binds to the antigen 63 bound to the immobilized antibody 62. In this case, since the enzyme-labeled antibody 65 is added in excess, a bound portion (hereinafter referred to as B) formed by binding of the antigen 83 and the antibody 65 is formed.
The free part that is not bound to (free1 below F
) can be done.

■ Therefore, by cleaning the free part (f
ree, F) discard the excess enzyme-labeled antibody.

That is, B/F separation is performed.

■ Next, perform an enzymatic reaction. Since the enzyme activity at this time is determined by the amount of enzyme-labeled antibody in the bound portion, the amount of enzyme-labeled antibody represents the amount of antigen bound to the immobilized antibody, that is, the amount of antigen to be measured.

In the competitive reaction method, as shown in FIG. 5, (1) An antibody is immobilized on a solid phase, and an antigen to be measured and a labeled antigen in which a label is bound to the same antigen as the antigen to be measured are added.

(2) As a result, an antigen-antibody reaction occurs, and the antigen to be measured and the labeled antigen bind to the immobilized antibody in proportion to their respective amounts.

(2), (2) Next, B/F separation is performed in the same manner as in the sandwich method, and an enzyme reaction is performed. Since the enzyme activity at this time is determined by the amount of enzyme-labeled antibody in the bound portion, the amount of the antigen to be measured can be determined from the amount of labeled antigen added using a calibration curve.

All of the above methods are so-called separation methods, and there are also non-separation methods that do not perform B/F separation.

Although the non-separation method is quick in measurement time and easy to operate, it has drawbacks such as low measurement sensitivity. In comparison, separation methods have high measurement sensitivity, but are extremely complex and cumbersome to operate, and are difficult to automate, conventionally being carried out manually in most cases.

When the above-mentioned immunoassay is automated, antibodies or antigens are immobilized on a solid phase such as polystyrene balls or glass beads, and the solid phase reagent is used as a solid phase reagent.

Since this solid phase reagent is unstable, the storage solution is usually placed in a grooved container, and when performing enzyme immunoassay, the solid phase reagent is poured one by one from the container into the reaction detection container. The sample is transferred, the sample is dispensed, the labeling reagent is dispensed, B/F separation is performed, and washing is performed, and then the bound portion is transferred to the detector.

By the way, when measuring using an enzymatic reaction, for example, a dye solution is formed and the reaction is detected by measuring the color density with a colorimeter, but the detection sensitivity is low, so the immune reaction is difficult to detect. The chemiluminescence of the product is detected by injecting a luminescent reagent into the product. And for chemiluminescence detection,
There are two types: a flow cell type that measures the reaction product by continuously flowing it, and a batch type that measures each test tube using test tubes. Luminescence detection is also performed by placing a detection element such as a photomultiplier close to the luminescent reaction solution.

[Problem to be solved by the invention]

In automated immunoassays that detect such chemiluminescence,
After the final B/F separation fist washing, if the luminescent reagent is injected directly into the solid phase reagent and luminescence detection is performed, the amount of luminescent light detected may be low or the amount of luminescent light detected may not be constant. There is.

That is, there was a problem that data could not be collected stably.

The present invention has been made in view of the above circumstances, and is an automatic immunoassay device that uses chemiluminescence to measure immune reactions. The present invention relates to an automatic immunoassay device that can collect data.

[Means to solve the problem]

To this end, the present invention provides an automatic immunoassay device that measures an immune reaction by injecting a luminescent reagent into an immune reaction product in a reaction tube and causing chemiluminescence, in which a luminescent auxiliary reagent is added before the luminescent reagent is injected into the reaction tube. It is characterized by providing means for injecting.

[Effect]

The automatic immunoassay device of the present invention can prepare the conditions for the chemical reaction related to luminescence before injecting the luminescent reagent by injecting a predetermined luminescent capture reagent before injecting the luminescent reagent into the reaction tube, and further Since the particulate solid phase reagent can be uniformly dispersed in the liquid, the luminescent reaction when the luminescent reagent is injected can occur stably and smoothly, and the stability of data can be maintained.

〔Example〕

Examples will be described below based on the drawings.

FIG. 1 is a diagram showing an embodiment of the configuration of an automatic immunoassay device according to the present invention, and FIG. 2 is a diagram showing an embodiment of a cartridge used in the automatic immunoassay device according to the present invention. In the figure, 1 is a reaction turntable, 2 is a cartridge turntable, 3 is a reagent turntable, 4 is a sample turntable, 5 is a sample cup, 6 is a disbo chip,
7 to 9 are arm mechanisms, 10 is a detector, 11 is dust, 1
2 is a control processing chamber, 21 is a cartridge main body, 22 is a filter, 23 is a solid phase reagent, 24 is a discharge hole, 25 is an opening, 26 is an aluminum cap, 27 is an orifice, and 28 is a front part.

In FIG. 1, the cartridge turntable 2 is a table that stores and rotates disposable cartridges (reaction detection containers) as shown in FIG. 2, and is composed of ten removable cassettes. 30
An example is shown in which 2 cartridges can be stored. According to this, since cartridges of the same immobilized antibody are stored in each section, cartridges for 10 items can be prepared. The reagent turntable 3 is a rotating table that stores reagent bottles of labeled antibodies and disbo chips for dispensing the labeled antibodies, and an example is shown in which reagent bottles for 10 types, that is, 10 items can be stored. . The sample table 7 + - bull 4 is a rotating table that stores sample cups 5 containing samples and disbo chips 6 for dispensing the samples, and has two disbo chips 8 inside corresponding to each sample cup 5. An example is shown in which it is possible to store .

The reaction turntable 1 is used to perform sample dispensing, addition of labeled antibodies, stirring reaction by vibration, washing, etc., as described above, while the cartridge of the cartridge turntable 2 is set and rotated one position at a time. . The arm mechanisms 7 to 9 are mechanisms for inserting and removing cartridges and dispensing reagents and samples between the reaction turntable 1, cartridge turntable 2, reagent turntable 3, and sample turntable 4, respectively. The circle aXbs c shows the locus of . Further, the detector 10 is for injecting a luminescent reagent into the cartridge after the reaction and detects the amount of luminescence, and the dust 11 is for discarding the cartridge after the amount of luminescence has been detected.

As shown in FIG. 2, the cartridge used in the automatic immunoassay device according to the present invention has a cylindrical cartridge main body 2L, a solid phase reagent 23 is placed therein, and a filter 22 is placed below it.
Furthermore, a narrow discharge hole 24 is provided halfway below the filter 22, and an opening 25 at the upper end is closed with an aluminum cap 26. Solid phase reagent 2
3 has antibodies immobilized on the surface of granules with a diameter of several tens of μm. Since the antibodies and antigens in solid phase reagent 23 are proteins, they are easily decomposed, so they are treated with preservatives and buffers to maintain a constant pH. It is immersed in a preservation solution consisting of liquid, etc.

Further, there is an orimeter 27 cut in the part where the ejection hole 24 is provided, and by bending the tip end 28 at the orimeter 27, the cartridge can be easily removed from the cartridge body 21, and the ejection hole 24 can be passed through. be able to. The discharge hole 24 is formed with an extremely small diameter, and
Since the filter 22 is disposed, when the cartridge is used, the tip 28 is attached to the cartridge body 21.
Even when removed from the drain hole 24, dispensed samples, reagents, washing water, etc. are not easily discharged from the drain hole 24.
By supplying pressurized air from the opening 25 at the upper end,
Alternatively, it can be discharged by suctioning through the discharge hole 24.

Therefore, the cartridge has an aluminum cap 2 at the top end.
6, the solid phase reagent 23 is stored on the filter 22 with its lower end completely sealed by the tip 28, and is stored in the cartridge turntable 2. and,
When this cartridge is transferred from the cartridge turntable 2 to the reaction turntable 1 by the arm mechanism 7, the tip part 28 is removed in the dust 11, and the storage solution is discharged and washed at the next position of the reaction turntable 1. That's what I do.

Next, the operation of the automatic immunoassay device according to the present invention will be explained based on the flow system.

Figure 3 is the overall flow diagram, 31 is a drain tank,
32 is a compressor, 33 is an in-out switching valve, 34-37, 51 and 53 are pumps, 38-41 are tanks, 42 is a three-way joint, 43 is a resistance pipe, 44 is a preheater, 45, 52 and 54 are valves, 4B indicates a mixer.

As shown in FIG. 3, the flow system is set up on a reaction turntable 1 with 29 positions. The flow system for dispensing samples and reagents, washing, etc. is connected to the base point. First, the cartridge is set on the reaction turntable at the base point Posisilon 2, and the reaction turntable is alternately rotated in two predetermined positions.

The cartridge after the reaction is transferred to the detector 10.

First, each flow system connected to the reaction turntable 1 will be explained in FIG.

The drain tank 31 is for storing the storage solution and cleaning solution discarded from each cartridge of the reaction turntable 1, and is connected to a drain path provided in an annular shape below each position of the reaction turntable 1. . The compressor 32 is for supplying pressurized air for discarding the storage solution, cleaning immediately after that by washing Tl, B/F separation, disposing of the sample or reagent remaining at the tip of the disposable chip, and for cleaning.

The tank 38 is for storing a luminescent auxiliary reagent, and the tanks 39 and 40 are for storing a luminescent reagent, respectively.The in-out switching valve 33 and the pumps 34 to 37 are for transporting the luminescent auxiliary reagent, diluent, and luminescent reagent. It is something. A buffer solution stored in the tank 41 is used as the diluting solution and the washing solution. As this buffer solution, a mixture of a surfactant, sugar, etc. that promotes the immune reaction is used. Details of these injection methods will be described later.

In the cleaning process, the valve 45 is selectively opened and closed, and the buffer solution is injected from the buffer 41 through the resistor tube 43, the preheater 44, and the valve 45 into the cartridges of the positions ①, [phase], and 0, respectively. Then, the air valves are selectively opened and closed, and pressurized air is supplied from the compressor 32 to the cartridges of positions θ, o, and 6 through the air valves. In normal cleaning, injection of a cleaning solution (buffer solution) and disposal using pressurized air are performed four times.

In Bogiscene 2, the sample dispensing flow system and the labeled antibody reagent dispensing flow system are connected so that the sandwich method and competitive reaction method can be applied, but these are respectively separated from the sample cup or reagent bottle. It is inhaled and dispensed using a disposable tip.

In this case, sample or reagent remains on the tip, so
A pressurized air flow system is connected to blow them out with pressurized air. In the sample dispensing system, this is the flow system of the pulse 52. The tip of the disposable tip is inserted into the sample cup of the sample turntable 4, and the sample is aspirated by the sampling pump 51.
After dispensing into the cartridge, the system is switched to this flow system.

Similarly, when dispensing a reagent, insert the tip of the disbo tip into the reagent bottle of the reagent turntable 3,
After inhalation and dispensing with the reagent pump 53, the valve 54 switches to a pressurized air flow system. Additionally, since the amount of suction becomes unstable when the internal pressure increases, a valve for opening to the atmosphere is also provided.

Next, the rotation of the position of the reaction turntable 1 will be explained.

The horizontal arm machine 7 operates with the position button ① to transport a new cartridge from the cartridge turntable 2, remove the tip, and set it.

Even if the tip 28 shown in Fig. 2 is removed from the cartridge when setting a new cartridge in the positive position ■, the storage solution inside will not be disposed of. and discard the storage solution in the cartridge.

At the next positioning (2), the cleaning valve is set at the upper end opening of the cartridge, and cleaning is performed by repeatedly sending cleaning liquid and pressurized air alternately, for example, four times.

Next, add the diluent using 1 liter of Bodine. This is to make it easier to cause an immune reaction, since when blood, serum, urine, etc. are directly injected, various components may interfere with the immune reaction.

Then, aspirate the sample from the sampling cup with Vodicillin ■ using a disposable tip and dispense it into the cartridge.

After that, the immune reaction (first reaction) is promoted by applying vibration and stirring each time it rotates one position at a time, and by repeating water supply and drainage with pressurized air four times at position @, the cleaning process is performed first. Perform the B/F separation as described.

Here, we will explain the operation after B/F separation in detail.
First, rotate the body σ in the forward direction for 20 degrees, stop at position ■, inject the diluent (buffer) as a buffer, and then rotate the body 1 degree forward in the forward direction to increase the position by 1 position from the previous time. Proceed to the next point ■. After stirring by vibration, 2
Rotate in the forward direction at the 0 position, stop once with Bodicilon ■, and dispense the labeled antibody. The diluent has the effect of stabilizing the reaction temperature by preheating, facilitating and promoting the immune reaction, and enhances the stirring effect when the next labeling reagent is dispensed. This is the operation in the case of the Sanderutsch method.

That is, by alternately performing an operation of rotating the pitch of the 10th position and an operation of rotating the pitch of the 20th position, the position is advanced one position at a time from the previous position. In this way, even in the sandwich method, it is possible to adopt an apparatus configuration in which the labeling reagent is dispensed at the same time as sample dispensing. As a result, immunoassays can be performed using the same flow system even in the competitive reaction method by controlling the rotation operation so that the sample and labeled antigen are simultaneously dispensed in the body cylinder (2).

After that, the immune reaction (second reaction) is promoted by stirring and applying vibration while rotating alternately at a pitch of 10 positions and a pitch of 20 positions as in the first reaction up to position 1. Then, B/F separation by washing is carried out again at Bojishi Nippon Chemical O.

Then, at the starting point 0, a luminescence auxiliary reagent for intensifying the luminescence is added, and at the first starting point 1, the cartridge is transferred to the detector 10. The detector 10 measures the amount of luminescence immediately after adding the luminescence reagent to the cartridge.

In the above operation, for example, it stands still at position 1 x ■ for 12 seconds, rotates in the forward direction at a pitch of 20 positions, stands still at position position O for 12 seconds, and then rotates at a pitch of 10 positions until position position ■. The cartridge is rotated and advances one position at a time from position ■ to position ■ for 24 seconds. At position position ■, the cartridge is first transferred to detector ] 0 in 12 seconds, and a new The cartridge is brought from the cartridge turntable 2 and set. In this case, the first reaction takes about 3 minutes, the second reaction takes about 5 minutes, and the immune reaction measurement for one sample can be performed in about 10 minutes in total, which is about 150 tests.
A measurement speed of hr can be achieved.

When measuring multiple items with one sample, in position 1, solid phase reagent cartridges corresponding to each measurement item are set on the cartridge turntable, and the same sample is dispensed into those cartridges. Then, reagents corresponding to the measurement items are dispensed.

Then, the amount of light emitted is measured by the detector 10, and the process ends.

If you need to measure again, use the remaining Disbochip to dispense the sample. To this end, the sample turntable shown in FIG. 1 stores two disbo chips corresponding to each sample. Re-measurement is required not only when the measured value shows an abnormal value that significantly deviates from the preset range, but also when a judgment value is given for each measurement item in advance as a primary screening, and the next measurement item is determined based on the judgment result. may be set. For example, in a serum hepatitis test, first a test for HBS antigen is performed, and if the result is positive, a test for HBE or HBS antigen is performed. In addition, the reason why a disposable disbo tip is used is that the range is very wide in the case of immunoassay, and conventional pipettes cannot clean it completely.

Next, a method for injecting a luminescent reagent will be described in detail.

As mentioned above, the tank 38 contains the luminescence auxiliary reagent, and the tank 3
Reference numerals 9 and 40 each house a luminescent reagent, and send the luminescent auxiliary reagent, diluent, and luminescent reagent through the in-out switching valve 33 and pumps 34 to 37. A buffer solution stored in the tank 41 is used as the diluting solution and the washing solution. As this buffer solution, a mixture of a surfactant, sugar, etc. that promotes the immune reaction is used. The liquid tank 41 has a sealed structure and is configured to supply pressurized air from the compressor 32 and send liquid by applying pressure.
The cleaning liquid is controlled to have a stable predetermined temperature and flow rate through a resistance tube 43, a preheater 44, and a valve 45, thereby facilitating the reaction and stabilizing the reaction.

Similarly, for luminescent reagents, by adding a heater to the mixer 46, it is possible to stabilize the temperature during the luminescent reaction.

Incidentally, the luminescent reagent varies depending on the labeling substance, but for example, in the case of acridinium, a mixture of hydrogen peroxide and alkali, luminol (1,
B1nol), a mixture of hydrogen peroxide and Fe ions, dioxetone (1.2-[) loxetoo
In the case of e), a mixture of hydrogen peroxide and a fluorescent material is used, but since these react in a short time, just before detecting the luminescence, a mixture of hydrogen peroxide and a fluorescent material is used. injected at the same time.

Since the luminescent reagent is mixed immediately before detection in this manner, the luminescent reagent can be maintained in a stable state.

Further, as the luminescence auxiliary reagent, when the labeling substance is the above-mentioned acridinium, a hydrochloric acid acidic solution such as dilute hydrochloric acid can be used. That is, when the luminescent reagent is injected, acidic P
By preparing the H condition S, the luminescent reaction of acridinium can be ensured, and at the same time, by uniformly dispersing the solid phase reagent in the form of particulates in the luminescent auxiliary reagent solution, contact between acridinium and the luminescent reagent can be achieved. The combination of these two effects enables stable data collection.

In the above embodiment, position 1 of the reaction turntable 1
Although we have explained how to dispense the luminescent auxiliary reagent in ``O", the dispensing of the luminescent auxiliary reagent can be done as long as it is done before dispensing the luminescent reagent into the cartridge, so it is not limited to this. isn't it. That is, a separate boat may be provided on the way to transfer the cartridge from the position I O to the detector 10, and the luminescent auxiliary reagent may be injected there, or the luminescent reagent may be injected within the detector after being transferred to the detector 10. The luminescent auxiliary reagent may be dispensed prior to dispensing. In any case, it goes without saying that for the purpose of uniformly dispersing the solid phase reagent in the luminescence auxiliary reagent solution, it is preferable to stir the cartridge after injecting the luminescence auxiliary reagent.

Further, in the above embodiment, the mixing of two reagents was explained, but it may be one reagent or three or more reagents. In addition, if two or more types of samples exist in the same cartridge, separate dispensing nozzles are prepared to detect them separately, and each reagent can be detected individually by injecting each reagent at a different time. can.

Furthermore, when there is one type of measurement target and other background, the first luminescent reagent is dispensed to cause the background substances other than the target substance to emit light, and then the second reagent is dispensed. Note that it is also possible to detect the luminescence of the target luminescent substance, which allows background to be removed.

〔Effect of the invention〕

As described above, according to the present invention, by injecting a predetermined luminescent auxiliary reagent before injecting a luminescent reagent into a reaction tube, it is possible to prepare the condin of the chemical reaction related to luminescence before injecting the luminescent reagent, and By uniformly dispersing the particulate solid phase reagent in the liquid, preparations can be made for smooth contact between the labeling substance and the luminescent reagent, ensuring a reliable and smooth luminescent reaction when the luminescent reagent is injected. This allows for stable data collection.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the configuration of an embodiment of an automatic immunoassay device according to the present invention, FIG. 2 is a diagram showing an embodiment of a cartridge used in the automatic immunoassay device according to the present invention, and FIG. The overall flow diagram, FIG. 4 is a diagram for explaining the principle of measurement by the sandwich method, and FIG. 5 is a diagram for explaining the principle of measurement by the competitive method. 1... Reaction turntable, 2... Cartridge turntable, 3... Reagent turntable, 4...
Sample turntable, 5...Sample cup, 6
...Disbo chip, 7-9...Arm mechanism, 10
...detector, 11...dust, 12...control processing section, 21...cartridge body, 22...filter 23...solid phase reagent, 24...discharge hole, 25...
- Opening, 26... Aluminum cap, 27... Orimeter, 28... Front pant part. Applicant: JEOL Ltd.

Claims (1)

[Claims] (1) In an automatic immunoassay device that measures an immune reaction by injecting a luminescent reagent into an immune reaction product in a reaction tube and causing chemiluminescence, a means for injecting a luminescent auxiliary reagent before injecting the luminescent reagent into the reaction tube. An automatic immunoassay device comprising: